Transgenic animals expressing chimeric antibodies for use in preparing human antibodies

ABSTRACT

The invention provides transgene constructs for expressing chimeric antibodies, and transgenic non-human host animals carrying such constructs, wherein the chimeric antibodies comprise human variable regions and constant regions of the non-human transgenic host animal. The presence of immunoglobulin constant regions of the host animal allows for generation of improved antibodies in such transgenic host animals. Subsequently, the chimeric antibodies can be readily converted to fully human antibodies using recombinant DNA techniques. Thus, the invention provides compositions and methods for generating human antibodies in which chimeric antibodies raised in vivo in transgenic mice are used as intermediates and then converted to fully human antibodies in vitro.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase of International Application No.PCT/U.S.07/008,231, filed Mar. 30, 2007, which claims priority to U.S.Provisional Application Ser. No. 60/744,104 filed on Mar. 31, 2006, thecontents of which are hereby incorporated in their entirety. Thespecification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Sep. 30, 2008.

BACKGROUND OF THE INVENTION

Antibodies have proven to be effective therapeutic agents in humans forthe treatment of a wide variety of disorders, including cancer,autoimmune diseases and infectious diseases. Although originally mousemonoclonal antibodies were tried as therapeutic agents, they generallyproved to be unsuitable for use in humans due to the occurrence of ahuman anti-mouse antibody (HAMA) response. Rather, antibodies composedin part or entirely of human antibody amino acid sequences currently arethe antibody agents of choice for use in humans. Of the numerousantibodies approved by the FDA for use in humans or currently inclinical trials, certain antibodies contain mouse variable regionslinked to human constant regions and typically are referred to aschimeric antibodies. Others contain mouse CDRs within human frameworkand constant regions and typically are referred to as humanizedantibodies. Still others are composed entirely of human-derivedsequences (i.e., fully human variable and constant regions) andtypically are referred to as human antibodies.

A number of approaches are known in the art for preparing humanantibodies. In one type of approach, a library of human immunoglobulinsequences is screened on a display system (e.g., bacteriophage) with anantigen of interest to select antibody sequences having the desiredantigenic specificity (see e.g., U.S. Pat. Nos. 5,223,409; 5,403,484;and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty etal.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;6,582,915 and 6,593,081 to Griffiths et al.). Since this approach iscarried out in vitro, the human antibody sequences do not undergoaffinity maturation or somatic mutation during the selection process,which may result in antibodies of lower affinity as compared toantibodies generated in vivo.

Thus, in another type of approach, mice whose genomes have been modifiedto contain human immunoglobulin sequences are used to raiseantigen-specific antibodies by immunization with an antigen of interest.Such mice carry unrearranged human immunoglobulin genes (variable andconstant regions) on transgenes and/or transchromosomes, which genesundergo apparently normal rearrangement and isotype switching in themice. Moreover, somatic mutation occur during the maturation of theantibody response in these mice.

One example of such a mouse is the HuMAb Mouse® (Medarex, Inc.), whichcontains human immunoglobulin transgene miniloci that encodeunrearranged human heavy (μ and γ) and κ light chain immunoglobulinsequences, together with targeted mutations that inactivate theendogenous μ and κ chain loci (see e.g., Lonberg, et al. (1994) Nature368(6474): 856-859). Accordingly, the mice exhibit reduced expression ofmouse IgM or κ and, in response to immunization, the introduced humanheavy and light chain transgenes undergo class switching and somaticmutation to generate high affinity human IgGκ monoclonal (Lonberg, N. etal. (1994), supra; reviewed in Lonberg, N. (1994) Handbook ofExperimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995)Intern. Rev. Immunol. 13: 65-93 and Harding, F. and Lonberg, N. (1995)Ann. N.Y. Acad. Sci. 764:536-546). The preparation and use of HuMabmice, and the genomic modifications carried by such mice, are furtherdescribed in Taylor, L. et al. (1992) Nucleic Acids Research20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724;Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993)EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920;Taylor, L. et al. (1994) International Immunology 6: 579-591; andFishwild, D. et al. (1996) Nature Biotechnology 14: 845 851, thecontents of all of which are hereby specifically incorporated byreference in their entirety. See further, U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Pat.No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all toLonberg and Kay; and PCT Publication No. WO 01/14424 by Korman et al.

An alternative transgenic mouse system for expressing humanimmunoglobulin genes is referred to as the Xenomouse (Abgenix, Inc.) andis described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181;6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al. Like the HuMAbMouse® system, the Xenomouse system involves disruption of theendogenous mouse heavy and light chain genes and insertion into thegenome of the mouse transgenes carrying unrearranged human heavy andlight chain immunoglobulin loci that contain human variable and constantregion sequences.

Other systems known in the art for expressing human immunoglobulin genesinclude the KM Mouse® system, described in detail in PCT Publication WO02/43478 by Ishida et al., in which the mouse carries a human heavychain transchromosome and a human light chain transgene, and the TCmouse system, described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci.USA 97:722-727, in which the mouse carries both a human heavy chaintranschromosome and a human light chain transchromosome. In each ofthese systems, the transgenes and/or transchromosomes carried by themice comprise human immunoglobulin variable and constant regionsequences.

U.S. Pat. No. 6,596,541 provides a prophetic example of a homologousrecombinant mouse that expresses chimeric antibodies having humanvariable region sequences linked to mouse constant region sequences. Inthe example, the mouse heavy chain locus variable region (V-D-Jsegments) is precisely replaced with the human heavy chain V-D-Jcounterpart via a multi-step process. First, a large genomic fragment(greater than 20 kb) spanning the human immunoglobulin variable genesegments of interest is obtained and bacterial recombination is used toprepare a large targeting vector for use in eukaryotic cells (LTVEC)that includes homology arms totaling greater than 20 kb. The homologyarms contain sequences from the endogenous mouse immunoglobulin locus.The LTVEC is then introduced into mouse embryonic stem cells. A cell inwhich homologous recombination has occurred between the LTVEC and theendogenous mouse immunoglobulin locus is identified using a quantitativeassay to detect modification of allele (MOA) in the ES cells. No actualmice expressing chimeric antibodies, however, are described orcharacterized.

SUMMARY OF THE INVENTION

In this invention, transgenic animals expressing chimeric antibodies,comprising human variable regions and non-human constant regions, aredescribed and characterized. In particular, the constant regions arethose of the host animal (e.g., in mice, the constant regions are miceconstant regions). The animals of the invention can be made usingtransgenic microinjection technology and do not require the use ofhomologous recombination technology and thus are easier to prepare andselect than approaches using homologous recombination. Moreover, theexpression of the human Ig variable regions linked to the host animal Igconstant regions in the transgenic animals of the invention is thoughtto allow for improved trafficking and development of B cells andantibodies in vivo such that improved antibodies can be obtained inthese animals, as compared to transgenic animals that express human Igvariable regions linked to human Ig constant regions. Such improvementsin the antibodies can include, for example, increased somatic mutations,improved association with endogenous mouse accessory proteins, andimproved binding to mouse Fc receptors in vivo.

Moreover, the chimeric antibodies can readily be converted to fullyhuman antibodies by isolation of the sequences encoding the human Vregions and linkage of these sequences to human constant regionsequences using standard recombinant DNA technology in vitro. Thus, theinvention provides a means to obtain improved human antibodies, suitablefor use in therapy, through the use of a chimeric antibody intermediateraised in vivo in transgenic animals.

In the animals of the invention, a transgene comprising unrearrangedhuman immunoglobulin variable region sequences and at least one hostanimal constant region sequence (e.g., an IgM constant region) isprepared and inserted into the genome of the host animal (e.g., bypronuclear microinjection into a zygote of the host animal). Wheninserted into the genome of the host animal, the transgene constructundergoes rearrangement and expresses chimeric antibodies in thenon-human host animal, the chimeric antibodies comprising a humanvariable region and a constant region of the non-human host animal.Moreover, as demonstrated herein, the inserted transgene is capable ofundergoing trans-switching in the host animal with endogenous constantregions such that chimeric antibodies of different isotypes are obtainedin the animals.

Accordingly, in one aspect, the invention pertains to a transgeneconstruct comprising a plurality of unrearranged human immunoglobulin(Ig) variable region sequences operatively linked to at least oneimmunoglobulin (Ig) constant region sequence of a non-human host animal,wherein the transgene construct undergoes rearrangement in the non-humanhost animal and expresses chimeric antibodies in the non-human hostanimal. The chimeric antibodies comprise a human variable region and aconstant region of the non-human host animal. In a preferred embodiment,the plurality of unrearranged human Ig variable region sequences areheavy chain variable region sequences. Alternatively, the plurality ofunrearranged human Ig variable region sequences can be light chainvariable region sequences.

In a preferred embodiment, the construct comprises heavy chain variableregion sequences comprising V-D-J sequences. For example, the constructcan comprise, in 5′ to 3′ direction, a plurality of human V_(H) regions,a plurality of human D segments, a plurality of human J_(H) segments, aJ-μ enhancer from a non-human host animal, a μ switch region from anon-human host animal and a μ constant region from a non-human hostanimal. In one embodiment, the construct comprises four human V_(H)regions, 15 human D segments and six human J_(H) segments. A preferredtransgene construct is a 9B2 transgene construct.

As demonstrated herein, transgene constructs comprising human heavychain V-D-J sequences linked to a μ constant region of the non-humanhost animal are capable of undergoing trans-switching with an endogenousconstant region of the non-human host animal when the transgeneconstruct is integrated into the genome of the non-human host animalsuch that chimeric antibodies of more than one isotype can be raised inthe host animal. In a particularly preferred embodiment, the inventionprovides a transgene construct which comprises, in 5′ to 3′ direction, aplurality of human V_(H) regions, a plurality of human D segments, aplurality of human J_(H) segments, a mouse J-μ enhancer, a mouse μswitch region and a mouse μ constant region, wherein the transgeneconstruct, when integrated into a mouse genome, undergoestrans-switching with an endogenous mouse γ constant region such thatchimeric antibodies comprising human V regions and mouse constantregions of IgM and IgG isotype are produced in the mouse.

Although the presence of the μ constant region in the transgeneconstruct has been shown to be sufficient for trans-switching to occur,in certain embodiments it may be preferable to include more host animalconstant regions in the transgene construct itself, such that bothtrans-switching and cis-switching can occur to generate antibodies ofdifferent isotypes. Thus, in certain embodiments, the transgeneconstruct can include, for example, a γ constant region from thenon-human host animal or an cc constant region from the non-human hostanimal. Alternatively, the transgene construct can comprise all Igconstant regions of the non-human host animal (i.e., the transgeneconstruct comprises the entire constant region of the non-human hostanimal).

In another aspect, the invention pertains to transgenes for expressingchimeric antibodies in which the unrearranged human Ig variable regionsequences are human light chain variable region sequences, such as humankappa V-J sequences, linked to light chain constant region sequencesfrom the non-human host animal. For example, in one embodiment, theconstruct comprises, in 5′ to 3′ direction, a plurality of human V_(κ)regions, a plurality of human J_(κ) segments, a J-κ enhancer from anon-human host animal and a C_(κ) coding region from a non-human hostanimal.

Another aspect of the invention pertains to transgenic non-human hostanimals comprising one or more transgene constructs of the invention,wherein the animal expresses chimeric antibodies comprising human Igvariable regions and non-human host animal constant regions. Preferably,the transgene undergoes trans-switching and the animal expresseschimeric antibodies comprising human Ig variable regions and non-humanhost animal Ig constant regions of at least the IgM and IgG isotypes.Preferred non-human host animals are mice, although other animalssuitable for transgenesis are also encompassed by the invention.Moreover, preferably the endogenous immunoglobulin loci are inactivatedin the non-human host animal, for example by homologous recombination.In a preferred embodiment, an endogenous heavy chain locus of thetransgenic host animal is inactivated by disruption of the J_(H) region.In another preferred embodiment, an endogenous light chain locus of thetransgenic host animal is inactivated by disruption of the J_(κ) region.

Yet another aspect of the invention pertains to a method of making achimeric antibody specific for an antigen of interest. The methodcomprises immunizing a transgenic non-human host animal of theinvention, which comprises a transgene construct of the invention, withthe antigen of interest and obtaining from the animal a chimericantibody specific for the antigen of interest. For example, hybridomasexpressing chimeric antibodies can be prepared from the immunized hostanimal using standard techniques. In a preferred embodiment, the methodfurther comprises isolating from the animal nucleic acid encoding thechimeric antibody, replacing nucleic acid encoding the non-human hostanimal Ig constant region with nucleic acid encoding a human Ig constantregion to thereby convert the chimeric antibody to a human antibody andexpressing the human antibody. In certain embodiments, the humanantibody exhibits higher affinity toward the antigen of interest thanthe chimeric antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the 9B2 transgene construct and ofthe three fragments comprising the HCo26 transgene constructs alignedabove the mouse genomic immunoglobulin locus.

DETAILED DESCRIPTION OF THE INVENTION

This invention involves the use of a non-human transgenic animal thatexpresses chimeric antibodies as a host to raise a chimeric antibody toan antigen of interest, followed by conversion of the chimeric antibodyto a fully human antibody. The chimeric antibodies expressed in thetransgenic non-human host animal comprise human variable regions linkedto constant regions of the non-human host animal. The invention pertainsto transgene constructs, non-human transgenic host animals carrying suchtransgene constructs and methods of using such host animals to raisechimeric antibodies, which then can further be converted to fully humanantibodies.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the term “chimeric antibody” refers to an antibody inwhich at least one of the antibody chains (heavy or light) comprisesvariable regions sequences from one species (e.g., human) and constantregion sequences from another species (e.g., mouse). The term “chimericantibody” is intended to encompass antibodies in which: (i) the heavychain is chimeric but the light chain comprises V and C regions fromonly one species; (ii) the light chain is chimeric but the heavy chaincomprises V and C regions from only one species; and (iii) both theheavy chain and the light chain are chimeric.

As used herein, the term “transgene construct” refers to a nucleic acidpreparation suitable for introduction into the genome of a host animal.A “transgene construct” of the invention can comprise a single piece ofnucleic acid (such as the 9B2 transgene) or multiple pieces of nucleicacid (such as the HCo26 transgene). When the transgene constructcomprises multiple pieces of nucleic acid, the individual pieces makingup the transgene construct preparation contain overlapping sequencessuch that when they are introduced into the genome of the host animal,they recombine to create a contiguous transgene (see, for example, thefurther description of the HCo26 transgene herein).

As used herein, the term “isotype switching” refers to the phenomenon bywhich the class, or isotype, of an antibody changes from one Ig class toanother Ig class through a recombination process mediated by switchsequences.

As used herein, a “nonswitched isotype” refers to the isotypic class ofthe heavy chain that is produced when no isotype switching has takenplace. The C_(H) gene encoding the nonswitched isotype is typically thefirst C_(H) gene immediately downstream from the functionally rearrangedVDJ gene (e.g., the IgM isotype).

As used herein, the term “switch sequence” or “switch region” refers tothose DNA sequences, known in the art, that are responsible for switchrecombination resulting in Ig class switching.

As used herein, the term “trans-switching” refers to isotype switchingthat involves recombination between one switch region and another switchregion located on a different chromosome, such as recombination betweena transgene switch region and an endogenous switch region located on adifferent chromosome than the chromosome that harbors the transgene. Inparticular, it refers to recombination between a transgene switch regionand a switch region of the endogenous Ig constant region of thenon-human transgenic host animal.

As used herein, the term “cis-switching” refers to isotype switchingthat involves recombination between one switch region and another switchregion located on the same chromosome, such as recombination between twoswitch regions within a transgene or between two switch regions of theendogenous Ig locus, such as switch recombination between a μ constantregion and a γ constant region carried by the same transgene or by theendogenous Ig locus.

As used herein the term “unrearranged” or “germline configuration” inreference to an immunoglobulin V segment refers to the configurationwherein the V segment is not recombined so as to be immediately adjacentto a D or J segment.

As used herein, the term “rearranged” in reference to an immunoglobulinV segment refers to the configuration wherein the V segment ispositioned immediately adjacent to a D-J or J segment so as to encodeessentially a complete V_(H) or V_(L) domain, respectively.

As used herein, the term “a plurality of unrearranged immunoglobulin(Ig) variable region sequences” is intended to refer to constructs thatcontain more than one heavy or light chain variable region segment in anunrearranged configuration.

As used herein, the term “operatively linked” is intended to describethe configuration of a nucleic acid sequence that is placed into afunctional relationship with another nucleic acid sequence. For example,a promoter or enhancer is operatively linked to a coding sequence if itaffects the transcription of the sequence. With respect to the joiningof two protein coding regions, operatively linked means that the nucleicacid sequences being linked are contiguous and in reading frame. Forswitch sequences, operatively linked means that the sequences arecapable of effecting switch recombination.

I. Transgene Constructs for Expressing Chimeric Antibodies

The transgene constructs of the invention comprise a plurality ofunrearranged human immunoglobulin (Ig) variable region sequencesoperatively linked to at least one immunoglobulin (Ig) constant regionsequence of a non-human host animal, wherein the transgene constructundergoes rearrangement in the non-human host animal and expresseschimeric antibodies in the non-human host animal, the chimericantibodies comprising a human variable region and a constant region ofthe non-human host animal. In this context, the term “at least one Igconstant region sequence” is intended to mean a sequence encoding atleast one constant region isotype sequence, such as an IgM constantregion sequence or a kappa constant region sequence. The inventionencompasses transgene constructs encoding chimeric heavy chain sequencesor encoding chimeric light chain sequences. When integrated into anon-human transgenic host animal, the variable regions of the transgeneconstructs undergo rearrangement such that functional heavy chain orlight chain variable regions are created. Moreover, in certainembodiments, the integrated transgene construct undergoestrans-switching such that chimeric antibodies of more than one isotypeare made in the transgenic non-human host animal. When the transgeneconstruct encodes chimeric heavy chain sequences, it comprises at leastthe IgM constant region of the non-human host animal and may containadditional host constant regions although, as demonstrated herein, thepresence of the host IgM constant region alone, even without the known3′ IgH enhancers, is sufficient to secure maturation of the B cells andachieve trans-switching.

Accordingly, in one aspect, the plurality of unrearranged human Igvariable region sequences in the transgene construct are heavy chainvariable region sequences. In particular, such heavy chain constructstypically comprise unrearranged human heavy chain V-D-J sequences. Suchheavy chain constructs also contain at least one Ig constant regionsequence of a non-human host animal and typically also containregulatory sequences including enhancers and switch sequences. Thus, ina preferred embodiment, the transgene construct comprises, in 5′ to 3′direction, a plurality of human V_(H) regions, a plurality of human Dsegments, a plurality of human J_(H) segments, a J-1 enhancer from anon-human host animal, a μ switch region from a non-human host animaland a μ constant region from a non-human host animal. In a particularlypreferred embodiment, the construct comprises four human V_(H) regions,15 human D segments and six human J_(H) segments, as described furtherin the Examples. A preferred but non-limiting example of a heavy chainconstruct of the invention is the 9B2 transgene.

Preferably, the heavy chain construct of the invention is capable, whenintegrated into a non-human host animal genome, of undergoingtrans-switching with an endogenous constant region of the non-human hostanimal. As demonstrated herein, the presence in the transgene constructof the μ switch region and the μ constant region of the non-human hostanimal is sufficient to allow for trans-switching to occur between theintegrated transgene and endogenous constant region sequences in thehost animal. Thus, a heavy chain construct of the invention thatcomprises only one host animal constant region sequence, μ, cannevertheless lead to the generation of chimeric antibodies of multipleisotypes (e.g., IgM and IgG) in the host animal as a result oftrans-switching.

Thus, another preferred transgene construct of the invention comprises,in 5′ to 3′ direction, a plurality of human V_(H) regions, a pluralityof human D segments, a plurality of human J_(H) segments, a mouse J-μenhancer, a mouse μ switch region and a mouse μ constant region, whereinthe transgene construct, when integrated into a mouse genome, undergoestrans-switching with an endogenous mouse γ constant region such thatchimeric antibodies comprising human V regions and mouse constantregions of IgM and IgG isotype are produced in the mouse.

While the presence of the μ constant region (and associated switchsequences) alone may be sufficient for the generation of multipleisotypes of chimeric antibodies, in certain instances it may bedesirable to include more than one host animal constant region sequencein the heavy chain transgene construct such that both trans-switchingand cis-switching can occur. Accordingly, in various embodiments, aheavy chain transgene construct of the invention may comprise one, two,three or more constant regions of the non-human host animal. Forexample, in another embodiment, in addition to the p constant region,the construct can further comprise a γ constant region (and associatedswitch sequences) from a non-human host animal. For example, when amouse is used as the transgenic non-human host animal, the heavy chaintransgene construct can include murine p coding sequences (andassociated switch sequences) and, additionally, can include one or moreof the murine γ1, γ 2a, γ 2b and γ 3 coding sequences (and associatedswitch sequences). In yet another embodiment, the construct can compriseall immunoglobulin (Ig) constant regions of the non-human host animal(i.e., the construct contains the entire C region of the non-human hostanimal, encompassing the μ, δ, γ, α, and ε constant sequences).

In another aspect, a transgene construct of the invention comprises aplurality of unrearranged human light chain variable region sequences,linked to a light chain constant region sequence of the non-humantransgenic host animal. In particular, such light chain constructstypically comprise unrearranged human light chain V-J sequences, eitherkappa V-J sequences or lambda V-J sequences. In a preferred embodiment,the chimeric construct is a chimeric kappa light chain constructcomprising, in 5′ to 3′ direction, a plurality of human V_(κ) regions, aplurality of human J, segments, a J-κ enhancer from a non-human hostanimal (e.g., the mouse κ internal enhancer) and a C_(κ) coding regionfrom a non-human host animal.

In another embodiment, the chimeric light chain construct can be alambda light chain construct. In both the mouse and human Ig loci, thereis a cluster of multiple V_(λ), genes followed by repeated clusters ofJ_(λ)-C_(λ) units. Thus, in one embodiment, a chimeric lambda chaintransgene can be constructed by linking a plurality of human V_(λ)regions to a plurality of human J_(λ)-non-human C_(λ) combination units.Alternatively, one can select a single human J_(λ)-non-human C_(λ)placed downstream of a plurality of human V_(λ) regions. Anotherpossible configuration is to place a plurality of human V_(λ) regionsupstream of the endogenous non-human J_(λ)-C_(λ) clusters, which wouldlead to chimeric antibodies comprising a human V_(λ) region linked to anon-human J_(λ) region and a non-human C_(λ) region.

The transgene constructs of the invention can be prepared using standardrecombinant DNA techniques. Cloning vectors containing polylinkers areuseful as starting vectors for insertion of DNA fragments of interest.Non-limiting examples of such suitable cloning vectors are described inthe Examples. Plasmids or other vectors (e.g., YACs) carrying humanunrearranged heavy chain or light chain immunoglobulin sequences havebeen described in the art (see e.g., U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; and U.S.Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6, 150,584 and 6,162,963, allto Kucherlapati et al.) Such plasmids and other vectors can be used asthe source of human unrearranged heavy chain or light chain variableregions to be included in the transgene constructs of the invention.Additionally or alternatively, suitable human Ig variable region DNA canbe obtained from genomic libraries using standard techniques. DNAencoding constant region sequences of the non-human host animal,including the coding sequences of the constant region and the associatedenhancer and switch regions, similarly can be obtained from genomiclibraries using standard techniques. For example, the B6 BAC library(Invitrogen) of murine genomic DNA can be used as the source of murineconstant region sequences for inclusion in the transgene constructs ofthe invention.

In addition to the variable and constant coding regions, cis-actingregulatory sequences typically are needed for chromatin accessibility,proper V(D)J recombination, class switching, high levels of antibodyexpression and other locus control functions. Promoter regions locatednear the V(D)J gene segments may play a role in chromatin accessibilityand V(D)J recombination. Intronic enhancers between the J_(H) and IgMcoding region and J_(κ) and kappa coding regions have been identified.Additionally, downstream 3′ DNase hypersensitivity regions have beenidentified, which make up the IgH locus control region (LCR). Classswitch recombination is dependent on promoters and sterile transcriptsof switch regions upstream of the different heavy chain constantregions. An origin of replication has also been identified downstream ofthe 3′ DNase hypersensitive sites which may demark a boundary for theIgH locus. Preferably, the constructs of the invention contain at leastthe promoter regions located near the V(D)J gene segments, one or moreoperative switch regions and an intronic enhancer. Inclusion of allgenomic DNA from the 5′ intronic enhancers through downstream 3′ LCRs,and possibly beyond, may be preferred (but not essential) for highlevels of Ab generation, development and maturation.

The appropriate genomic DNA fragments from the human Ig variable regionsand from the non-human host animal Ig constant regions are thenoperatively linked through ligation into a cloning vector, followed bycharacterization of the vector (e.g., by restriction fragment analysisor sequencing or the like) to ensure proper arrangement of the genomicfragments. A non-limiting example of the creation of a heavy chaintransgene construct of the invention is described in detail in Example1.

To prepare the transgene construct for microinjection or other techniquefor transgenesis, the transgene construct can be isolated from thevector in which it is carried by cleavage with appropriate restrictionenzymes to release the transgene construct fragment. The fragment can beisolated using standard techniques, such as by pulse field gelelectrophoresis on an agarose gel, followed by isolation of the fragmentfrom the agarose gel, such as by β-agarase digestion or byelectroelution. For example, the agarose gel slice containing thetransgene construct fragment can be excised from the gel and the agarosecan be digested with β-agarase (e.g., from Takara), using standardmethodology.

II. Preparation and Characterization of Transgenic Non-Human HostAnimals

Another aspect of the invention pertains to a transgenic non-human hostanimal that comprises one or more of the transgene constructs of theinvention (i.e., the transgene construct(s) is integrated into thegenome of the host animal), such that the animal expresses chimericantibodies comprising human Ig variable regions and non-human hostanimal Ig constant regions. Preferably, the transgene constructundergoes trans-switching and the animal expresses chimeric antibodiescomprising human Ig variable regions and non-human host animal Igconstant regions of at least IgM and IgG isotypes.

The transgenic non-human host animals of the invention are preparedusing standard methods known in the art for introducing exogenousnucleic acid into the genome of a non-human animal. Preferred non-humananimals are mice, although other animal species that are (i) suitablefor transgenesis and (ii) capable of rearranging immunoglobulin genesegments to produce an antibody response may also be used. Examples ofsuch species include but are not limited to rats, rabbits, chickens,goats, pigs, sheep and cows.

A preferred method for preparing the transgenic non-human animal, inparticular a transgenic mouse, is that of pronuclear microinjection.This technology has been known for over twenty years and is wellestablished (see e.g., Wagner, T. E. et al. (1981) Proc. Natl. Acad.Sci. USA 78:6376-6380; U.S. Pat. No. 4,873,191 by Wagner and Hoppe). Ingeneral, the method involves introducing exogenous genetic material intothe pronucleus of a mammalian zygote (e.g., mouse zygote) bymicroinjection to obtain a genetically transformed zygote and thentransplanting the genetically transformed zygote into a pseudopregnantfemale animal. The embryo is then allowed to develop to term and thegenome of the resultant offspring is analyzed for the presence of thetransgenic material. Southern blot analysis, PCR or other such techniquefor analyzing genomic DNA is used to detect the presence of a uniquenucleic acid fragment that would not be present in the non-transgenicanimal but would be present in the transgenic animal. Selective breedingof transgenic offspring allows for homozygosity of the transgene to beachieved.

Although the preferred embodiment of the invention comprises transgenicmice prepared by pronuclear microinjection, the invention encompassesother non-human host animals, including but not limited to rats,rabbits, pigs, goats, sheep, cows and chickens. Techniques for creatingtransgenic animals of each of these species have been described in theart.

For example, preparation of transgenic rats is described in Tesson, L.et al. (2005) Transgenic Res. 14:531-546, including by techniques suchas DNA microinjection, lentiviral vector mediated DNA transfer intoearly embryos and sperm-mediated transgenesis. Methods of transgenesisin rats are also described in Mullin, L. J. et al. (2002) Methods Mol.Biol. 180:255-270.

Preparation of transgenic rabbits is described in, for example, Fan, J.et al. (1999) Pathol. Int. 49:583-594; Fan, J. and Watanabe, T. (2000)J. Atheroscler. Thromb. 7:26-32; Bosze, Z. et al. (2003) Transgenic Res.12:541-553.

Preparation of transgenic pigs is described in, for example, Zhou, C. Y.et al. (2002) Xenotransplantation 9:183-190; Vodicka, P. et al. (2005)Ann. N.Y. Acad. Sci. 1049:161-171. Alternative transgenesis techniquesto pronuclear microinjection in pigs include adenovirus mediatedintroduction of DNA into pig sperm (see e.g., Farre, L. et al. (1999)Mol. Reprod. Dev. 53:149-158) and linker-based sperm-mediated genetransfer (Chang, K. et al. (2002) BMC Biotechnol. 2:5).

Preparation of transgenic goats is described in, for example, Ebert, K.M. et al. (1991) Biotechnology (NY) 9:835-838; Baldassarre, H. et al.(2004) Reprod. Fertil. Dev. 16:465-470. Somatic cell nuclear transfer ingoats is described in, for example, Behboodi, E. et al. (2004)Transgenic Res. 13:215-224.

Preparation of transgenic sheep is described in, for example, Ward, K.A. and Brown, B. W. (1998) Reprod. Fertil. Dev. 10:659-665; Gou, K. M.et al. (2002) Shi Yan Sheng Wu Xue Bao 35:103-108

Preparation of transgenic cows is described in, for example, Donovan, D.M. et al. (2005) Transgenic Res. 14:563-567. Gene transfection of donorcells for nuclear transfer of bovine embryos is described in, forexample, Lee S. L. et al. (2005) Mol. Reprod. Dev. 72:191-200.

The state of the art in the preparation of transgenic domestic farmanimals is also reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech.24:285-298.

Preparation of transgenic chickens is described in, for example, Pain,B. et al. (1999) Cells Tissues Organs 165:212-219; Lillico, S. G. et al.(2005) Drug Discov. Today 10:191-196. Use of retroviral vectors in thepreparation of transgenic chickens is described in, for example, Ishii,Y. et al. (2004) Dev. Dyn. 229:630-642.

The transgenic non-human animals of the invention may comprise an Igheavy chain transgene construct for expressing chimeric antibodies or anIg light chain transgene construct for expressing chimeric antibodies,or both a light and a heavy chain construct for expressing chimericantibodies. Typically, to create animals that carry more than onetransgene, animals carrying individual transgenes are prepared and thencross-bred to create animals carrying more than one transgene. Animalsthat inherit both transgenes can be identified and selected by standardtechniques for analysis of genomic DNA in the animals. Moreover, ananimal of the invention carrying a transgene construct for expressingone chimeric Ig chain (e.g., a chimeric heavy chain) can be cross-bredwith an animal that carries a transgene that expresses a non-chimericform of the other Ig chain (e.g., a fully human light chain). In suchanimals, the antibodies expressed comprise one chimeric chain (e.g., achimeric heavy chain) and one non-chimeric chain (e.g., a non-chimeric,fully human light chain). Such animals are also encompassed by theinvention and suitable for use in raising chimeric antibodies against anantigen of interest. See the Examples for a further description of atransgenic animal expressing a chimeric heavy chain transgene constructand a fully human light chain transgene construct, wherein the chimericheavy chain transgene construct undergoes trans-switching to produceantibodies of multiple isotypes (e.g., IgM and IgG) in the animals.

The non-human transgenic host animals of the invention express chimericantibodies, in which at least one chain of the antibody comprises ahuman variable region sequence and a constant region sequence of thehost animal. The non-human transgenic host animals of the invention donot express any fully human antibodies (comprising both a fully humanheavy chain and a fully human light chain) since they do not carry bothhuman constant region sequences (human C_(H) and human C_(L)) in theirgenome. Thus, the antibody repertoire of these animals differs from thatof the HuMab Mouse® (e.g., as described in PCT Publication WO 94/25585).Although the Ig transgenes in the HuMab Mouse® are described in WO94/25585 as being capable of undergoing trans-switching with endogenousmouse Ig constant regions to create chimeric antibodies, the Igtransgenes in the HuMab Mouse® contain human variable region sequencesand human constant region sequences and thus, in addition to possiblyexpressing chimeric antibodies, these mice also express a repertoire offully human antibodies. In contrast, the transgenic non-human hostanimals of the present invention only express a repertoire of chimericantibodies, without the additional presence of fully human antibodies inthe animals.

Furthermore, the presence of constant regions of the non-human hostanimal in the chimeric antibodies expressed by the host animals isthought to allow for improved B cell and antibody development in vivo,as compared to transgenic animals that express antibodies havingconstant regions of another species (e.g., human constant regions). Forexample, the chimeric antibodies that contain host animal Fc regions arethought to associate better with endogenous host animal accessoryproteins (e.g., Igα/Igβ or other signaling molecules) for more naturalreceptor signaling leading to more normal B cell development and higherantibody production. Moreover, the chimeric antibodies are thought tobind better to host Fc receptors, leading to increased recirculation andantigen presentation and, thus, more normal immune responses. Stillfurther, the presence in the transgene construct of host animalregulatory sequences in the host animal-derived constant region isthought to lead to improved genetic regulation of antibody expression.Thus, such an environment in which there is more normal B celldevelopment, more serum antibodies, improved genetic regulation ofantibody expression and better germinal center formation should lead toa more diverse, and possibly higher affinity, antibody population withappropriate somatic mutations.

Thus, in certain embodiments, chimeric antibodies raised in thenon-human transgenic host animals of the invention may exhibit increasedsomatic mutations as compared to fully human antibodies raised innon-human transgenic host animals. The presence of a constant region ofthe non-human host animal in the chimeric antibodies of the inventioncan also afford the advantage that the antibody may possess additionaleffector functions (e.g., ADCC, complement fixation) in the host animalspecies that would not be present with antibodies having human constantregions. Thus, a chimeric antibody raised according to the invention maybe amenable for use in particular animal models of disease in which afully human antibody may not be suitable for use. For example, chimericantibodies raised according to the invention that comprise murineconstant regions may be amenable for use in mouse models of disease thatinvolve murine effector functions mediated by the murine Fc region.

In a preferred embodiment, a non-human transgenic host animal of theinvention has one or more of its endogenous immunoglobulin lociinactivated. It is preferable that the endogenous Ig loci be inactivatedso that endogenous host animal antibodies are not expressed in theanimal together with the expression of the chimeric antibodies. Inparticular, inactivation of the endogenous Ig loci prevents interferencefrom endogenous antibodies and simplifies the detection of the chimericantibodies. Accordingly, in one embodiment, a transgenic host animal(e.g., mouse) of the invention has at least one endogenous heavy chainlocus inactivated and more preferably has both endogenous heavy chainloci inactivated. Additionally or alternatively, the transgenic hostanimal (e.g., mouse) of the invention has at least one endogenous lightchain locus inactivated and more preferably has both alleles of thekappa loci or the lambda loci, or both, inactivated. In the mostpreferred embodiment, the host animal has a homozygous inactivation ofthe endogenous Ig heavy chain locus and a homozygous inactivation of theendogenous Ig kappa light chain locus. When the kappa locus isinactivated, it is not essential to disrupt the lambda locus; however,if a chimeric lambda light chain transgene is to be used, it isdesirable to inactivate the endogenous lambda locus.

The endogenous Ig loci are preferably inactivated by homologousrecombination using a targeting vector that inserts an exogenoussequence into the endogenous Ig locus such that expression of theendogenous Ig genes is disrupted. Preferred regions for insertion arethe J_(H) and J_(κ) regions. Once a host animal having a heterozygousdisruption of a heavy chain or light chain locus is obtained, the animalcan be bred to homozygosity for the disruption by standard breedingtechniques. A mouse strain having a homozygous disruption of theendogenous heavy chain locus at J_(H) has been previously described inthe art (see e.g., Examples 10 and 11 of U.S. Pat. No. 5,545,806), ashas a mouse strain having a homozygous disruption of the endogenouskappa light chain locus at J_(κ) and C_(κ) (see e.g., Example 9 of U.S.Pat. No. 5,545,806). Such mice can be used as breeding partners withmice carrying one or more transgenes of the invention to achieve a mousestrain carrying one or more transgenes and having its endogenous Ig lociinactivated. Such mouse strains are described in further detail in theExamples.

III. Preparation of Chimeric Antibodies in Transgenic Non-Human Animals

Another aspect of the invention pertains to method of making a chimericantibody specific for an antigen of interest. The method comprisesimmunizing a transgenic non-human host animal of the invention with theantigen of interest and obtaining from the animal a chimeric antibodyspecific for the antigen of interest.

Thus, to prepare chimeric antibodies in a transgenic non-human animal ofthe invention, first the animal is immunized with an antigen ofinterest. For example, immunization techniques that have previously beenused to raise fully human antibodies in transgenic mice carrying humanIg heavy and light chain transgenes (such as the HuMab Mouse® or theXenomouse) can similarly be used to raise antibodies in the animals ofthe invention. Immunization techniques previously used in the HuMabMouse® are described in, for example, Lonberg, N. et al. (1994) Nature368:856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851and PCT Publications WO 98/24884 and WO 01/14424. Preferably, mice are6-16 weeks of age upon the first infusion of antigen and a purified orrecombinant preparation of antigen (e.g., 5-50 μg) is used to immunizethe mice intraperitoneally. Typically, multiple animals (e.g., between 6and 24 animals) are immunized for each antigen.

Cumulative experience with various antigens in the HuMab Mouses haveshown that the transgenic mice respond well when initially immunizedintraperitoneally with antigen in complete Freund's adjuvant, followedby every other week IP immunizations (up to a total of 6) with antigenin incomplete Freund's adjuvant. However, adjuvants other than Freund'shave also been found to be effective and can be used additionally oralternatively. The immune response can be monitored over the course ofthe immunization protocol with plasma samples being obtained byretroorbital bleeds. The plasma can be screened by ELISA and mice withsufficient titers against the antigen of interest can be used to preparemonoclonal antibodies. Mice can be boosted intravenously with antigenthree days before sacrifice and removal of the spleen and/or lymphnodes.

Chimeric antibodies, as a polyclonal mixture, can be obtained from thehost animal or, more preferably, monoclonal antibodies can be preparedusing B cells obtained from the host animal. Monoclonal antibodies canbe prepared and selected by one of a variety of suitable methods knownin the art including, but not limited to, (i) hybridoma generation(discussed farther below), (ii) PCR amplification of antibody genesdirectly from B cells of obtained from the host animal (see e.g.,Babcook, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848)and (iii) phage display of an antibody library prepared from B cells ofthe host animal, followed by screening of the phage display library fora monoclonal antibody of interest (see e.g., PCT Publication WO01/25492)

In a preferred embodiment, hybridomas producing chimeric monoclonalantibodies of the invention are generated. To generate such hybridomas,splenocytes and/or lymph node cells from immunized mice can be isolatedand fused to an appropriate immortalized cell line, such as a mousemyeloma cell line (e.g., P3×63-Ag8.653 (ATCC, CRL-1580) or SP2/0 (ATCC,CRL-1581)). Cells can be fused using techniques well established in theart, such as chemically-mediated fusion (e.g., with PEG) orelectrofusion. Once extensive hybridoma growth has occurred, individualwells can be screened by ELISA For example, in animals expressing achimeric Ig heavy chain transgene and a fully human kappa light chaintransgene, wells can be screened by ELISA for expression of antibodiescomprising mouse IgM and human kappa or mouse IgG and human kappa. Usingsimilar techniques, the supernatants can be tested for the presence ofmouse IgG that binds to the antigen used for immunization. Hybridomaspositive for an antibody of interest can be subcloned at least twice bylimiting dilution. The stable subclones can then be cultured in vitro togenerate antibody material in tissue culture medium for furthercharacterization.

IV. Conversion of Chimeric Antibodies to Fully Human Antibodies

In the methods of the invention, once a chimeric antibody of interesthas been raised in the transgenic non-human host animal, the method canfurther comprise isolating from the animal, or a B cell from the animal,a nucleic acid encoding the chimeric antibody and replacing nucleic acidencoding the non-human host animal Ig constant region with nucleic acidencoding a human Ig constant region to thereby convert the chimericantibody to a human antibody and expressing the human antibody.

Thus, once a chimeric antibody of interest has been identified, it canbe converted to a fully human antibody using standard recombinant DNAtechniques. For example, DNA encoding the variable region (light orheavy chain) from the chimeric antibody can be obtained by standardmolecular biology techniques (e.g., PCR amplification or cDNA cloningusing a hybridoma that expresses the antibody of interest) and the DNAcan be inserted into an expression vector such that the variable regionsequences are operatively linked to a human constant region sequence, aswell as to transcriptional and translational control sequences. Anantibody heavy chain gene and an antibody light chain gene can beinserted into separate vectors or, more typically, both genes areinserted into the same expression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). An expression vector can be used thatalready encodes heavy chain constant and light chain constant regions ofthe desired isotype such that the V_(H) segment is operatively linked tothe C_(H) segment(s) within the vector and the V_(L) segment isoperatively linked to the C_(L) segment within the vector. Anon-limiting examples of suitable expression vectors for expressingfully human antibodies the pIE family of vectors as described in U.S.Patent Application No. 20050153394 by Black. Preferred constant regionisotypes present in the expression vectors include human IgG1 and IgG4constant regions for the heavy chain and the human kappa constant regionfor the light chain.

In the context of antibody expression vectors, the term “operativelylinked” is intended to mean that an antibody variable region is ligatedinto the expression vector such that the coding sequences of thevariable region are in-frame with the coding sequences of the constantregion. Moreover, the variable and constant regions are positionedwithin the vector such that the transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. Additionally, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors carry regulatory sequences that control the expression of theantibody chain genes in a host cell. The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the antibody chain genes. Such regulatory sequencesare described, for example, in Goeddel (Gene Expression Technology.Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Itwill be appreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences, maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV), SimianVirus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter)and polyoma. Alternatively, nonviral regulatory sequences may be used,such as the ubiquitin promoter or βglobin promoter. Still further,regulatory elements can be composed of sequences from different sources,such as the SRα promoter system, which contains sequences from the SV40early promoter and the long terminal repeat of human T cell leukemiavirus type 1 (Takebe, Y. et al (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Preferred selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

Other sequences that can be included in the expression vector includethose that enhance expression of the antibody genes in stabletransfectants, such as sequences that alter chromatin structure toprevent silencing of the transfected gene. A preferred example is a UCOE(ubiquitous chromatin opening element), which can enhance expression oftransfected sequences irrespective of their site of integration in astable transfectant.

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies in either prokaryotic or eukaryotichost cells, expression of antibodies in eukaryotic cells, and mostpreferably mammalian host cells, is the most preferred because sucheukaryotic cells, and in particular mammalian cells, are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Prokaryotic expression of antibodygenes has been reported to be ineffective for production of high yieldsof active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

The conversion of a chimeric antibody of the invention to a fully humanantibody is described in further detail in Example 6. As demonstrated inthat example, conversion of the chimeric form to the fully human formmaintained the binding properties of the antibody toward its targetantigen. In certain instances (such as in Example 6), the fully humanform of the antibody may even exhibit improved binding toward itstarget, such as higher affinity toward its target than the chimericform. Thus, in certain embodiments of the method for converting thechimeric antibody to a human antibody, the resultant human antibodyexhibits higher affinity toward the antigen of interest than theoriginal chimeric antibody.

EXAMPLES

In the following examples, certain plasmids, homologous recombinant miceand transgenic mice that have been previously described were used asstarting materials to create additional transgenes and transgenic mousestrains.

Plasmids and DNA Clones:

The pGP1 and pGP2 are general cloning vectors whose construction isdescribed in U.S. Pat. No. 5,545,806 (see in particular Example 4 andFIGS. 7 and 8). Both are pBR322-derived plasmids that have been modifiedto contain large polylinkers. In pGP1, the polylinker is flanked by rarecutting NotI sites for building large inserts. pGP2 was derived frompGP1 and includes an additional SfiI site located between the MluI andSpeI sites in the polylinker. pGP1b and pGP2b are similar pBR322-derivedcloning vectors. The construction of pGP1b also is described in U.S.Pat. No. 5,545,806 (see, in particular, Example 12), whereas theconstruction of pGP2b is described in U.S. Pat. No. 5,625,126 (see, inparticular, Example 36 and FIGS. 77A and 77B).

The pHC2 plasmid also is described in U.S. Pat. No. 5,545,806 (see inparticular Example 12 and FIGS. 25 and 31). pHC2 contains fourfunctional human immunoglobulin heavy chain variable (V_(H)) regions, 15human D segments, all six human J_(H) segments, the human J-μ enhancer,human μ switch region, all of the human μ coding exons, and the human γ1constant region, including the associated switch region and steriletranscript associated exons, together with 4 kb flanking sequencingupstream of the sterile transcript initiation site contained on a NotIfragment.

The RP23-109B20 (B20) genomic C57BL/6J mouse DNA clone was obtained fromInvitrogen and contains mouse J_(H) regions through mouse IgG2a codingregions. The RP23-116C22 (C22) genomic C57BL/6J mouse DNA clone was alsoobtained from Invitrogen and contains mouse IgG2b coding regions andapproximately 200 kb downstream of the mouse IgG2b coding regions.

Homologous Recombinant Mice:

Mice in which the endogenous immunoglobulin heavy chain locus and/or theendogenous immunoglobulin light chain locus have been disrupted byhomologous recombination have been previously described. In theseexamples, mice were used in which: (i) the murine light chain J_(κ)regions were deleted and replaced with the neo^(R) gene (referred toherein as the “JKD” genotype); (ii) the murine heavy chain Cμ region wasdisrupted by insertion of the neo^(R) gene in the opposite reading frameof the Cμ gene (referred to herein as the “CMD” genotype); and/or (iii)the murine heavy chain J_(H) region was deleted and replaced with theneo^(R) gene (referred to herein as the “JHD” genotype). Construction ofmice carrying the JHD modification is described in Examples 10 and 11 ofU.S. Pat. No. 5,545,806. Construction of mice carrying the CMDmodification is described in Example 1 of U.S. Application No.20020086014. Construction of mice carrying the JKD modification isdescribed in Example 9 of U.S. Pat. No. 5,545,806.

Transgenic Mice:

Mice carrying an unrearranged human light chain immunoglobulintransgene, comprising multiple V_(κ) regions, J_(κ) regions and theentire human kappa constant region, have been described previously. Inthese examples, mice were used that carry the KCo5 light chaintransgene, which was created by co-injection of a human kappa lightchain minilocus and a YAC clone comprising multiple human V_(κ)segments. Construction and characterization of mice strains carrying theKCo5 light chain transgene are described in detail in Example 38 of U.S.Pat. No. 6,255,458. Through breeding with the homologous recombinantmice strains described above, additional mice strains were obtained inwhich (i) the KCo5 transgene was present (KCo5+); (ii) the endogenouslight chain gene had been disrupted (JKD+/+) and (iii) the endogenousheavy chain gene had been disrupted (either JHD+/+ or CMD+/+).

Example 1 Construction of a Chimeric Transgene For Expressing ChimericAntibodies Comprising Human V_(H) and Mouse C_(H) Regions

A transgene construct was prepared that contained unrearranged humanheavy chain VDJ segments linked to the mouse Jμ enhancer region, μswitch region and μ coding region, as follows.

Intermediate Vectors

The intermediate vector pGP2-1 was constructed by digesting thepreviously described pGP2b with NotI, releasing the polylinker andligating with a synthetic linker constructed by annealed overlappingoligos DMTM12 (5′-GGCCGCACGCGTGTC GACTC-3′) (SEQ ID NO: 1) and DMTM13(5′-GCCGAGTCGACACGCGTGC-3′) (SEQ ID NO: 2). The resulting pGP2-1 plasmidthen has a polylinker with a NotI site followed by a MluI, and a SalIsite. The orientation and linker sequence were confirmed by sequencing.

The intermediate vector pIM-m2 was constructed by digesting thepreviously described pGP1b with NotI, releasing the polylinker andligating with a synthetic linker constructed by annealed overlappingoligos DMTM39 (5′-GGCCGCATTCGCCGG CTAACGGCGCCTA TAACGAGTTC-3′) (SEQ IDNO: 3) and DMTM40 (5′-GGCCGAACGGCTTATAGGCGCCG TTAGCCGGCGAATGC-3′) (SEQID NO: 4). The resulting pIM-m2 plasmid then has a polylinker with aNotI site followed by a NgoMIV site, a NarI site, a MluI site.

The intermediate vector pIM-m3 was constructed by digesting thepreviously described pGP1b with XhoI and HindIII, releasing part of thepolylinker and ligating with a synthetic linker constructed by annealedoverlapping oligos DMTM37 (5′-TCGAGGCCGGCATGATAG GCGCCGTCGACA-3′) (SEQID NO: 5) and DMTM38 (5′-AGCTTGTCGACGGCGCCTA TCATGCCGGCC-3′) (SEQ ID NO:6). The resulting pIM-m3 plasmid then has a polylinker with these sites:NotI-XhoI-NgoMIV-NarI-Sall-HindlIl-NotI.

Construction of Chimeric Transgene

pHC2 has a unique MluI restriction site located downstream of the most3′ human J_(H) segment and 5′ of the human J-μ enhancer. Theapproximately 44 kb NotI-MluI fragment from pHC2, containing fourfunctional human variable regions, 15 human D segments and all six humanJ_(H) segments, was isolated and cloned into the intermediate vectorpGP2-1. The new plasmid (phVDJ2) was screened by observing anapproximate 44 kb fragment released by NotI and MluI digestion andsouthern blot hybridization to a probe just 5′ of the human J-μenhancer.

To isolate the mouse J-μ enhancer, mouse μ switch region, all of themouse μ coding regions and the mouse 8 coding regions, the B20 BAC wasdigested with NgoMIV and NarI. The resulting 40 kb fragment was isolatedby pulse field gel electrophoresis (PFGE) and cloned into pIM-m2. Theresulting plasmid (pIM-m2-mED) was screened by the appearance of a PCRfragment using primers specific for mouse IgM and by observing anapproximate 40 kb fragment released by NgoMIV and NarI digestion.Furthermore, the mouse μ switch region was checked by southern blot tobe full length (as compared to the starting B20 BAC) as part of thisregion often becomes deleted.

To isolate the mouse J-μenhancer, mouse μ switch region, all of themouse μ coding regions, pIM-m2-mED was digested with XhoI (located 3′ ofthe mouse IgM coding region) and NarI and ligated with a syntheticlinker constructed by annealed overlapping oligos DMTM72(5′-TCGACTCCGCGGTTTAAACTGG-3′) (SEQ ID NO: 7) and DMTM73 (5′-GGCGCCAGTTTAAACCGCGGAG-3′) (SEQ ID NO: 8). The new resulting plasmid(pIM-m2-mEM) contained the mouse J-μ enhancer, mouse μ switch region,all of the mouse μ coding regions on an approximate 13 kb NgoMIV-NarIfragment with no internal XhoI or SalI sites. pIM-m2-mEM was screened bythe appearance of a PCR fragment using DMTM73 and DMTM76, which isspecific for a region 5′ of the XhoI site and points downstream towardsthe newly inserted linker.

The approximate 13 kb NgoMIV-NarI fragment, containing the mouse J-μenhancer, mouse μ switch region and all of the mouse μ coding regions,was then cloned into pIM-m3 to create pIM-m3-mEM, which adds a 5′ XhoIsite and a 3′ SalI site to the fragment. pIM-m3-mEM was screened byobserving an 13 kb fragment released by NgoMIV and NarI digestion and byXhoI and SalI digestion.

The final phVDJ2-mEM construct was then constructed by ligating the 13kb XhoI-SalI fragment, containing the mouse J-p enhancer, mouse μ switchregion and all of the mouse 1 coding regions, from pIM-m3-mEM into theSalI site 3′ of the human VDJ region in phVDJ2. Clones were checked fordirectional cloning by the production of a PRC product from DMTM79(5′-GCTGGAAAGAGAACTGTCGGAGTGGG-3′) (SEQ ID NO: 9), which anneals justdownstream of the human J_(H) region pointing downstream, and DMTM80(5′-CCAAAGTCCC TATCCCATCATCCAGGG-3′) (SEQ ID NO: 10), which anneals tothe mouse J-μ enhancer and points upstream. Furthermore, the final cloneof phVDJ2-mEM (called 9B2, illustrated schematically in FIG. 1) waschecked by southern blot to contain the full length mouse μ switchregion (as compared to the starting B20 BAC). The final construct thuscontains the human VDJ regions of pHC2 upstream of the mouse J-μenhancer, mouse μ switch region, and all of the mouse μ coding regionson an approximately 57 kb fragment.

Example 2 Preparation and Screening of Transgenic Mice

The approximately 57 kb NotI-SalI fragment from clone 9B2 of phVDJ-mEM(described in Example 1) was released from the vector and isolated byPFGE. An agarose gel slice with the 9B2 insert was excised and theagarose was digested with β-agarase (Takara) according to themanufacturer's protocol. The 9B2 fragment was micro-injected (bystandard methods) into fertilized oocytes. DNA was injected into F1 miceof wild type (JHD−/− CMD−/−, JKD−/−, KCo5−)×KCo5 mice (JHD−/−, CMD+/+,JKD+/+, KCo5+/+). Potential founder mice were screened for the 9B2transgene by PCR with DMTM79 and DMTM80 using tail DNA as template.There were 4 resulting founder mice (9B2-52, 9B2-56, 9B2-58, and 9B2-65)on the JHD−/−, CMD+/−, JKD+/−, KCo5+/− strain background.

Since mice of the CMD+/− genotype still carry the endogenous mouse heavychain J region, it was desirable to breed these founder mice with micecarrying the JHD deletion to obtain mice of the genotype CMD −/−,JHD+/+. Thus, founder mice positive for the 9B2 transgene were then bredto JHD (KCo5) mice (JHD+/+, CMD−/−, JKD+/+, KCo5+/+ mice) and genotypedfor 9B2, JHD, CMD, JKD and KCo5.

To generate 9B2 transgenic animals on the appropriate strain background:(9B2+, JHD+/+(CMD−/−), JKD+/+, KCo5+), founder offspring were selectedin part by their genotype (how similar they were to the final desiredstrain configuration) and if they were shown to have any pre-immunemouse IgG, or seemingly elevated post immunization mouse IgG levels.Offspring that carried either JHD+/+(knock out for mouse J_(H) region)or JHD+/− and CMD+/− (functionally knocked out for mouse heavy chainproduction) on a JKD+/+ and KCo5+ background were pre-immune titered fortotal mouse IgG/human kappa and mouse IgM/human kappa. The mice werethen challenged with Tetanus Toxiod (TT) at 1-2 week intervals, with 50μg of TT and 25 μg of Keyhole Limpet Hemocyanin (KLH) in 100 μl totalvolume of RIBI adjuvant, and titered for mouse IgG/human kappa, mouseIgM/human kappa and TT specific mouse IgG levels 10 days post the finalimmunization. Breeding continued for each founder line in this manneruntil 9B2+, JHD+/+ (CMD−/−) JKD+/+ strains were achieved.

Four founder lines carrying the 9B2 transgene were achieved, 9B2-52,9B2-56, 9B2-58 and 9B2-65. Each 9B2 founder line transmits the transgenein a Mendelian manner and the transgene is not linked to the sexchromosomes, or any obvious physical coat coloration genes.

Example 3 Characterization of Transgenic Mice Expressing ChimericAntibodies

In this example, the antibody responses of the four transgenic micefounder lines described in Example 2 to tetanus toxoid (TT) andInterferon-α (IFN-a) were examined to identify mice expressing highlevels of antibodies in which a human kappa light chain was paired witha heavy chain having a mouse IgG constant region, indicating that theheavy chain used in the antibody was derived from the transgeneconstruct that had undergone rearrangement and trans-switching with theendogenous mouse IgG constant region.

Tetanus Toxoid Responses

The antibody responses to TT for the four 9B2 mouse lines were examinedas follows. Five or six mice of each strain, consisting of four to fivetransgenic positive mice and at least one non-transgenic (ntg) mouse foruse as a negative control, were challenged weekly with TT at 50 μg of TTin 100 μl total volume of RIBI adjuvant for four weeks. Sera weretitered for mouse IgG/human kappa, mouse IgM/human kappa and TT-specificmouse IgG levels 10 days after the final immunization. Other controlswere the HC2 HuMab strain (carrying a fully human heavy chain transgene,pHC2 where the human V_(H), D and J_(H) gene segments are the same as inthe 9B2, and a fully human light chain transgene) and the B6 wild-typemouse strain.

Table 1 below summarizes the titer levels for each mouse within thecohort, along with the non-transgenic controls, the HC2 strain and theB6 strain. The results shown for the 9B2 strains are thepre-immunization (naive) serum levels of total mouse IgG/human kappaantibodies in μg/ml (column 3), the preimmune levels of total mouseIgM/human kappa antibodies in μg/ml (column 4), the post-immunizationserum levels of total mouse IgG/human kappa antibodies in μg/ml (column5), the post-immunization serum levels of total mouse IgM/human kappaantibodies at lowest titer dilution at 3× background (column 6) and TTspecific/mouse gamma antibodies in μg/ml (column 7). The appropriateanalogous results are also shown for the HC2 and B6 strains.

TABLE 1 Serum Titers of TT Immunized Transgenic Mice post 4 IMSpreimmune hK/mM mG/hK hK/mM mG/hK (titer- TT/mG Line Mouse ID# (μg/ml)(μg/ml) (μg/ml) 3xbkgd) (μg/ml) 9B2-52 98683 5.2 14.5 21.3 1620 7.798684 0.6 9.8 2.6 1620 0.3 98929 1.9 54.3 40.5 14580 34.6 98930 1.4 21.516.1 4860 0.0 98931 0.0 17.0 0.2 4860 0.0 99746-ntg 0.0 0.0 0.0 0 0.09B2-56 92986 0.2 317.7 0.5 43740 0.0 93382 0.4 199.3 1.3 43740 0.0 986931.6 164.5 2.4 43740 0.3 98694 0.4 183.7 0.2 43740 0.2 98695 0.2 138.40.5 14580 24.3 98697-ntg 0.0 0.5 0.0 0 0.0 9B2-58 96339 1.9 103.9 56.814580 48.1 94744 0.6 94.9 6.9 14580 13.6 96042 0.9 230.0 15.0 43740 4.697942 1.7 194.9 93.2 43740 24.2 97944 2.5 137.4 163.9 43740 67.797945-ntg 0.0 0.0 0.0 0 0.0 9B2-65 99770 1.1 11.8 3.5 4860 0.0 99771 6.011.8 21.2 4860 0.5 99775 18.9 18.7 36.0 60 0.3 99777 2.3 21.7 5.6 16200.0 99776 0.1 0.0 0.0 0 0.0 99778-ntg 0.2 0.0 0.0 0 0.0 hG/hk hk/hMhG/hk hk/hM TT/hG (μg/ml) (titer) (μg/ml) (titer) (μg/ml) HC2 98679100.5 1620 56.9 1920 0.2 98680 31.4 1620 57.1 4860 2.4 98681 23.6 162084.5 4860 0.6 mG/mK mK/mM mG/mK mK/mM TT/mG (titer) (titer) (titer)(titer) (μg/ml) B6 99898 180 43740 14580 43740 72712.1 99899 180 4374014580 43740 147832.0 99900 180 43740 14580 43740 38433.8

As expected, non-transgenic mice from each line do not express any mouseIgM/human kappa or mouse IgG/human kappa antibodies pre or postimmunization. In the transgenic mice, expression of mouse IgM/humankappa antibodies is believed to be a result of rearrangement of thetransgenic human VDJ segments to form a functional V region and splicingto the downstream transgenic mouse IgM constant region. Furthermore, inthe transgenic mice, mouse IgG/human kappa antibodies are believed to betrans-switched antibodies containing rearranged transgenic human VDJregions trans-switched to endogenous mouse IgG constant regions.

As the results in Table 1 demonstrate, lines 9B2-52 and 9B2-56 have lowlevels of naïve mouse IgG (trans-switched antibodies) and mouse IgM(derived from the transgene), whereas lines 9B2-58 and 9B2-65 containedhigher levels of mouse IgG antibodies in naïve serum. Furthermore, lines9B2-58 and 9B2-65 had elevated serum titers of mouse IgG/human kappaantibodies after immunization. All the mice tested for line 9B2-58expressed TT-specific mouse IgG, whereas the results were more variablefor the other lines, with some of the tested mice expressing TT specificmouse IgG post immunization and others not. The HC2 HuMab strain alsoshowed variability in the TT-specific responses of individual mice. Insome mice, levels of TT specific mouse IgG in sera were higher thanlevels of total mouse IgG/human kappa in sera. It is thought that thismay represent sera containing TT specific mouse IgG paired with anendogenous mouse lambda light chain.

Splenocytes from one TT immunized 9B2-58 mouse were fused viaelectrofusion and TT specific hybridomas were produced. The hybridomaswere stable and 12 hybridomas making anti-TT antibodies were initiallyisolated. Further cDNA sequence, protein characterization and BIACOREanalysis were performed on antibodies from nine of the hybridomas.Supernatants from the hybridomas were used in a BIACORE experiment todetermine affinity to TT, as well as on-rates and off-rates. Forcomparison, a fully human anti-TT antibody (raised in a HuMab Mouse® andexpressed in CHO cells) was used (referred to as TT hu IgG), as well asa recombinantly-produced chimeric mouse IgG/human kappa antibody(expressed in CHO cells) which contains the same human VDJ and humankappa chain as the TT hu IgG. Results of the affinity, on-rate andoff-rate comparison are shown in Table 2 below. Hybridoma clone namesare in column 1 while BIACORE data are in columns 2-4.

TABLE 2 BIACORE Analysis of Antibodies from TT Immunized Transgenic MiceAffinity On-rate Off-rate Clone Name (nM) (1/Ms) × 10⁴ (1/s) × 10⁴43H7C10 22.4 3.9 8.8 43C4E8 23.1 4 9.2 41A7B2 29 3.8 1.1 7G7A9 141 3.346 14D6G4 100 3.4 34 24F10A8 122 3.3 40 40G8F2 134 3.2 43 50C1G4 139 2.940 49A1A6 293 0.18 5.4 hu anti-TT 52 1.9 9.7 chi anti-TT 82 2.1 17

This data demonstrates that the chimeric anti-TT antibodies raised inthe transgenic mice have comparable affinities, on-rates and off-ratesas the fully human anti-TT antibody and the recombinantly-createdchimeric antibody made from the fully human anti-TT antibody. In fact,several of the chimeric antibodies raised in the 9B2-58 mouse havehigher affinities than the one fully human anti-TT antibody studied.

IFN-α Responses

To examine the antibody responses of the transgenic mice to IFN-α, sixto thirteen mice (five to eleven transgenic mice from each strain, andat least one non-transgenic mouse for use as a negative control) werechallenged weekly with 20 μg of IFN-α in 100 μl total volume of RIBIadjuvant. Serum was titered for mouse IgG/human kappa, mouse IgM/humankappa and IFN-α-specific mouse IgG levels 10 days post four and sevenimmunizations.

Table 3 below summarizes the titer levels for each mouse within thecohort, along with the non-transgenic controls. The results shown arethe pre-immunization (naive) serum levels of total mouse IgG/human kappaantibodies in μg/ml (column 3), the preimmune levels of total mouseIgM/human kappa antibodies in μg/ml (column 4), the post-4 immunizationserum levels of total mouse IgG/human kappa antibodies in μg/ml (column5), the post-4 immunization serum levels of total mouse IgM/human kappaantibodies in μg/ml (column 6), IFN-α specific/mouse gamma antibodies atlowest titer dilution at 3× background (column 7), the post-7immunization serum levels of total mouse IgG/human kappa antibodies inμg/ml (column 8), the post-7 immunization serum levels of total mouseIgM/human kappa antibodies in μg/ml (column 9) and IFN-α specific/mousegamma antibodies at lowest titer dilution at 3× background (column 10).

TABLE 3 Serum Titers of IFN-α Immunized Transgenic Mice Preimmune post 4IM post 7IM Total Total Total Total INFA/mG Total Total INFA/mG mG/hKhK/mM mG/hK hK/mM (titer- mG/hK hK/mM (titer- line mouse (μg/ml) (μg/ml)(μg/ml) (μg/ml) 3Xbkgd) (μg/ml) (μg/ml) 3Xbkgd) 9B2-52 82441 1.6 107.44.2 130.3 60 11.2 117.7 0 82444 1.0 64.8 DM 84028 1.6 107.7 7.7 106.7540 8.2 47.6 0 84029 1.4 77.5 1.1 51.9 20 1.2 40.3 0 84184 6.8 25.1 35.854.7 1620 22.8 49.9 162 0 84030-ntg 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.09B2-56 87314 0.6 174.4 1.9 111.9 540.0 2.6 268.7 0.0 87315 0.0 139.2 0.280.1 180.0 0.2 238.1 0.0 87316 0.1 519.9 0.2 164.7 180.0 0.1 559.0 0.087320 0.0 162.2 0.2 169.6 0.0 0.3 344.0 0.0 87321 0.9 165.3 4.1 385.10.0 0.2 720.6 0.0 89425-ntg 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 9B2-58 787442.9 64.9 66.7 264.5 4860.0 178.4 47.4 437 40.0 78751 0.9 170.6 96.2326.9 4860.0 4.5 126.1 486 0.0 78754 5.3 132.6 9.6 381.4 1620.0 181.6494.5 145 80.0 81198 5.8 94.2 24.1 243.2 4860.0 16.9 168.4 437 40.080620 7.8 169.7 27.5 360.8 14580.0 444.5 1043.1 437 40.0 80623 2.8 139.417.2 368.3 4860.0 86.4 166.5 145 80.0 80624 9.8 133.6 323.6 380.043740.0 1694.6 45.9 437 40.0 80460 5.8 89.5 24.0 0.8 4860.0 DM 80461 3.1217.2 12.8 385.6 4860.0 45.6 679.7 145 80.0 80468 9.3 62.7 40.1 151.11620.0 176.1 115.5 437 40.0 80473 1.8 102.7 16.5 159.5 1620.0 38.3 368.7437 40.0 78752-ntg 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 80621-ntg 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 9B2-65 97143 1.0 12.1 5.1 5.8 0.0 not done 97955263.2 19.5 68.7 14.1 0.0 97957 27.7 15.4 72.8 12.5 0.0 97959 2899.0 19.91878.0 15.8 50.0 97961 136.3 29.1 32.8 18.1 100.0 97144-ntg 0.0 0.0 0.00.0 0 DM = dead mouse

Again as expected, non-transgenic (ntg) mice from each line do notexpress any mouse IgM/human kappa or mouse IgG/human kappa antibodiespre or post immunization. In the IFN-α immunized transgenic mice,expression of mouse IgM/human kappa antibodies is believed to be aresult of rearrangement of the transgenic human VDJ segments to form afunctional V region and splicing to the downstream transgenic mouse IgMconstant region. Furthermore, in the transgenic mice, mouse IgG/humankappa antibodies are believed to be trans-switched antibodies containingrearranged transgenic human VDJ regions trans-switched to endogenousmouse IgG constant regions.

Similar to the results for the TT responses, lines 9B2-52 and 9B2-56have low levels of naïve mouse IgG (trans-switched antibodies) and mouseIgM (derived from the transgene), whereas lines 9B2-58 and 9B2-65contained higher levels of mouse IgG antibodies in naïve serum.Furthermore, lines 9B2-58 and 9B2-65 had elevated serum titers of mouseIgG/human kappa after four immunizations. Again, in general, all mice ofline 9B2-58 expressed high IFN-U. specific mouse IgG, while only somemice of the others lines expressed IFN-U. specific mouse IgG postimmunization.

Example 4 Analysis of Somatic Mutations in Chimeric Antibodies

In this example, the number of somatic mutations that occurred inanti-TT chimeric antibodies from the 9B2-58 transgenic mouse strain wasanalyzed, as well as the number of somatic mutations that occurred inchimeric antibodies from HuMab mice (which express both chimeric andfully human antibodies).

9B2-58 Transgenic Mice

Based on cDNA sequencing, the antibodies made by the anti-TT hybridomasgenerated from immunization of 9B2-58 transgenic mice, as described indetail in Example 3 above, were further characterized for their V_(H)and J_(H) usage and for which heavy chain isotype was present.Additionally, the number of somatic mutations occurring at the DNA leveland at the amino acid level was determined for the V_(H) segment only(not the D or J_(H) segments). The results are summarized in Table 4below:

TABLE 4 Sequence Analysis of Antibodies from TT Immunized 9B2 Mice SM SMHC Clone Name V_(H) J_(H) (DNA) (AA) Isotype 43H7C10* 3-30.3 4 15 9mIgG2b 43C4E8* 3-30.3 4 18 8 mIgG2b 41A7B2* 3-30.3 4 15 8 mIgG2b 7G7A9**3-30.3 4 5 3 mIgG2b 14D6G4** 3-30.3 4 5 3 mIgG2b 24F10A8** 3-30.3 4 5 3mIgG2b 40G8F2** 3-30.3 4 5 3 mIgG2b 50C1G4 4-34 4 17 7 mIgG1 49A1A6 4-344 17 7 mIgG1 hu anti-TT 3-33 4b 13 10 h IgG1 chi anti-TT 3-33 4b 13 10mIgG2a SM = somatic mutations; HC = heavy chain *indicates antibodiesdetermined to share the same light chain **indicates antibodiesdetermined to share the same light and heavy chains

With regard to V_(H) region usage, it should be noted that although the9B2-58 transgenic strain comprises different V_(H) regions than those inthe HuMab mouse used to raise the human anti-TT antibody, the 3-30.3 and3-33 V_(H) regions are similar, differing by only 2 amino acids. Thus,for several of the hybridomas, TT immunization selected similar V_(H)regions in the 9B2-58 transgenic strain as the V_(H) region used in ahuman anti-TT antibody raised in the HuMab mouse.

With regard to heavy chain isotype determination, since the transgeneinserted into the 9B2-58 strain contains only the mouse IgM constantregion, all of the chimeric antibodies that contain a human variableregion and a mouse IgG2b or IgG1, as observed herein, were producedthrough trans-switching from the transgene to various endogenous mouseconstant regions.

With regard to somatic mutations, at the DNA level, several of theantibodies from the 9B2-58 transgenic strain displayed greater numbersof somatic mutations than the human anti-TT antibody from the HuMabmouse. Moreover, chimeric antibodies with higher affinities for TT alsocontained more somatic mutations than other anti-TT antibodiesidentified from the same transgenic mouse. Although at the amino acidlevel, the chimeric antibodies from the 9B2-58 transgenic strain did notexhibit more somatic mutations than the human anti-TT antibody from theHuMab mouse (due to the degeneracy of the genetic code), the increasednumber of somatic mutations observed at the DNA level for several of thechimeric antibodies derived from the 9B2-58 transgenic strain supportsthe position that expression in transgenic mice of chimeric antibodiesthat contain a host constant region can lead to increased somaticmutations rates in the chimeric antibodies.

HuMab Mice

The data described above for the 9B2-58 strain with regard to somaticmutations is consistent with observations that have been made aboutsomatic mutation rates in the HuMab mouse, which express fully humanantibodies but which also may express chimeric antibodies due totrans-switching between the human heavy chain transgene and theendogenous mouse constant region.

For example, HuMab mice (HCo12/7 and HCo12 mice) were immunized with acell surface receptor and the spleens were isolated and fused accordingto standard hybridoma techniques. Eight anti-receptor antibodies wereisolated from the fusion mixture and sequenced. Three of the antibodieswere chimeric in nature, consisting of human VDJ regions linked to amouse IgG2b constant region. The remaining five antibodies were fullyhuman antibodies. cDNA sequence analysis of all the heavy chain variableregions showed that the chimeric and human antibodies used differentvariable region recombinations, and the chimeric antibodies had highersomatic mutations at the DNA level than the fully human antibodies andcorrelating higher amino acid differences. As for the 9B2-58 micedescribed above, the somatic mutation quantitation was determined forthe V_(H) segment only (not the D or J_(H) segments). The results aresummarized below in Table 5:

TABLE 5 Sequence Analysis of Human and Chimeric Antibodies from HuMabMice SM SM HC Clone Name V_(H) D_(H) J_(H) (DNA) (AA) Isotype Chimeric 13-23 3-9 4a 16 12 mIgG2b Chimeric 2 3-23 3-9 4b 16 13 mIgG2b Chimeric 33-23 3-9 4b 16 12 mIgG2b Human 1 3-30.3 nd 4b 8 6 hIgG1 Human 2 3-30.3nd 4b 7 7 hIgG1 Human 3 3-33 3-10 3b 2 2 hIgG1 Human 4 3-33 7-27 4b 2 2hIgG1 Human 5 3-33 5-5 3b 6 4 hIgG1 nd = not determined, SM = somaticmutation; HC = heavy chain

In another set of experiments, HuMab mice (HCo12/7) immunized with asoluble cytokine and the spleens were isolated and fused according tostandard hybridoma techniques. Six anti-cytokine antibodies wereisolated from the fusion and sequenced. Five of the antibodies werechimeric in nature consisting of human VDJ regions linked to a mouseIgG2a and IgG2b constant region. The remaining antibody was fully humanantibodies. cDNA sequence analysis of all the heavy chain variableregions showed that the chimeric antibodies and human antibody usedsimilar and different variable region recombinations and that thechimeric antibodies again had higher somatic mutations at the DNA levelthan the fully human antibody and correlating higher amino aciddifferences. Furthermore, the chimeric antibody #5 had a higher blockingefficiency than the fully human antibody #1 in functional blockingassays. The results are summarized below in Table 6:

TABLE 6 Sequence Analysis of Human and Chimeric Antibodies from HuMabMice SM SM HC Clone Name V_(H) D_(H) J_(H) (DNA) (AA) Isotype Chimeric 13-30.3 nd 4b 21 13 mIgG2b Chimeric 2 3-30.3 7-27 4b 8 7 mIgG2a Chimeric3 1-69 nd 4b 9 6 mIgG2a Chimeric 4 1-69 nd 4b 9 6 mIgG2a Chimeric 5 1-69nd 4b 25 15 mIgG2a Human 1 1-69 nd 4b 7 7 hIgG1 nd = not determined, SM= somatic mutation; HC = heavy chain

Thus, the results from the experiments described above for HuMab miceimmunized with two different antigens support the position thatexpression in transgenic mice of chimeric antibodies, that contain ahost constant region, can lead to increased somatic mutations in thechimeric antibodies, as compared to fully human antibodies expressed inthe transgenic mice.

Example 5 Generation of a Second Transgene for Expression of ChimericAntibodies

In this example, another transgene for expressing chimeric antibodies inmice was created, referred to as HCo26. The HCo26 transgene differs fromthe 9B2 transgene described in Example 1 in that the 9B2 transgenecontains only the mouse IgM constant region whereas the HCo26 transgenecontains all of the mouse constant region coding sequences.

HCo26 consists of three imbricate DNA fragments that containunrearranged human V, D and J regions and the mouse IgH locus from the5′ Eu enhancer through the identified 3′ DNase hypersensitive sites andorigin of replication (Zhou, et al. (2002) Proc. Natl. Acad. Sci. USA99:13693-13698). The construction of the HCo26 transgene is illustratedschematically in FIG. 1.

The first of the three fragments is the approximately 57 kb NotI-SalIfragment from clone 9B2 of phVDJ-mEM (described in Example 1) containingunrearranged human heavy chain VDJ segments linked to the mouse J-μenhancer region, μ switch region and μ coding region. Another fragmentconsists of the approximately 140 kb NgoMIV-NotI fragment of thepreviously described B20 BAC, which contains the mouse J-μ enhancer,mouse μ constant coding regions and the intervening mouse genomicregions through the mouse γ2b coding constant region. As the mouse J-μenhancer and mouse μ switch regions make up part of the 9B2 fragment,these regions are part of the overlapping regions of 9B2 and the B20fragment. Another fragment consists of the approximately 180 kbNotI-NotI fragment of C22 BAC, which consists of the mouse γ2b codingconstant region and the intervening mouse genomic regions through theDNase hypersensitive sites 3′ of the mouse cc coding regions to theendogenous NotI site found in the C22 BAC fragment. The mouse γ2b codingregions make up the overlapping regions of the B20 and the C22 BACfragments.

The approximately 57 kb NotI-SalI fragment from clone 9B2 of phVDJ-mEMwas released from the vector, as was the approximately 140 kbNgoMIV-NotI fragment of the B20 BAC and the approximately 180 kbNotI-NotI fragment of C22 BAC. Each was then isolated by PFGE. Anagarose gel slice with each of the fragments was excised and the agarosewas digested with β-agarase (commercially obtained from Takara)according to the manufacturer's protocol. The three fragments were thenmixed to a 1:1:1 stoichiometry and the mixture is referred to as HCo26.The HCo26 DNA mixture was then micro-injected (by standard methods) intofertilized oocytes. DNA was injected into JHD (KCo5) (JHD+/+ CMD−/−,JKD+/+, KCo5+) mice. Potential founder mice were screened for the 9B2transgene by PCR with DMTM79 and DMTM80 using tail DNA as template. Twodifferent founder mice, HCo26-05 and HCo26-16, tested positive for the9B2 transgene on the JHD+/+, CMD−/−, JKD+/+, KCo5+ strain background.Furthermore, Southern Blot analysis of genomic DNA from the HCo26-05mouse had 2 positive hybridizing BamHI bands with an approximately 1 kbprobe at the 3′ end of the C22 fragment. One band represents the 3′ endof the endogenous mouse IgH locus, while the other represents theintegration of the C22 fragment into the mouse genome in a randommanner.

Founder mice positive for the HCo26 transgenes were then bred to JHD(KCo5) mice (JHD+/+, CMD−/−, JKD+/+, KCo5+/+ mice) and genotyped for9B2, JHD, CMD, JKD and KCo5. HCo26-05 founder transmitted the HCo26transgene to several offspring.

These HCo26 founders are crossed to JHD (KCo5) mice to generate distinctstable HCo26 lines. HCo26 positive mice are tested for pre-immune levelsof mouse IgM/human kappa and mouse IgG/human kappa as described in theexamples above. Furthermore, HCo26 mice can be immunized with TT orother antigens and titered for mouse IgG/human kappa and antigen+/mouseIgG levels as described in the examples above.

Example 6 Conversion of Chimeric Antibodies to Fully Human Antibodiesand Comparison Thereof

To convert a chimeric antibody to a fully human antibody, cDNA of thevariable regions (comprising the rearranged human VDJ segments) of theheavy chain and light chain are isolated and sequenced. Total RNA isobtained from hybridoma cell pellets secreting the desired antibody byutilizing Qiagen RNeasy Mini Kit. cDNA is then prepared using the 5′RACE protocol utilizing Clontech SMART RACE cDNA Amplification Kit.Variable regions of each antibody are then amplified using a 3′ primerspecific for the mouse constant region paired with the 5′ RACE universalprimer mix. PCR products containing the variable regions are then clonedinto the Invitrogen TOPO TA DNA sequencing vectors. Minipreped DNAsamples are prepared and DNA sequenced. DNA sequences are then trimmedto include only the variable region of the desired antibody. Variableregions of the antibody are then matched to the germline human V(D)Jregions used to generate the 9B2 mice to ensure they are of transgeneorigin and thus of human origin Variable regions are also compared withmouse variable regions to rule out any mouse derived variable regions.cDNA sequences are compared with the N-terminal amino acid sequencingand mass spec analysis of the desired antibody to ensure the correctcDNA sequence is obtained.

Once the correct cDNA sequence is obtained, primers are synthesized toindependently PCR the coding regions of the heavy and light chainvariable regions. Furthermore, appropriate restriction sites are addedin frame to the coding regions to allow for cloning of the codingvariable regions directly into expression vectors that already encodefor a human constant region. Thus, a variable region (light or heavychain) is inserted into an expression vector such that the appropriatevariable region sequences are operatively linked to the appropriatehuman constant region sequence, as well as to transcriptional andtranslational control sequences. An antibody heavy chain gene and anantibody light chain gene can be inserted into separate vectors or, moretypically, both genes are inserted into the same expression vector. Anexpression vector can be used that already encodes heavy chain constantand light chain constant regions of the desired isotype such that theV_(H) segment is operatively linked to the C_(H) segment(s) within thevector and the V_(L) segment is operatively linked to the C_(L) segmentwithin the vector. Non-limiting examples of suitable expression vectorsfor expressing fully human antibodies include the pIE family of vectorsas described in U.S. Patent Application No. 20050153394 by Black.

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. Preferred mammalian host cells for expressing therecombinant antibodies of the invention include Chinese Hamster Ovary(CHO cells), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

The general methodology described above for converting a chimericantibody to a fully human antibody was used to convert an anti-TT mAbraised in the 9B2-58 strain of transgenic mouse to a fully humanantibody. More specifically, the heavy chain and light chain variableregions of the anti-TT antibody 43H7C10 (described in Example 4) (achimeric antibody consisting of a human VDJ variable region operativelylinked to a mouse gamma 2b constant region and paired with a fully humanKappa light chain) were cloned into expression vectors such that thehuman heavy chain variable region and light chain variable regionsequences were operatively linked to sequences encoding a human gamma 1constant region and a human Kappa constant region, respectively, toallow for the preparation of a recombinant fully human 43H7C10 antibody.The 43H7C10 human heavy chain and light chain variable region sequencesalso were cloned into expression vectors such that they were operativelylinked to sequences encoding the mouse gamma 2a constant region andhuman Kappa constant regions, respectively, to allow for the preparationof a recombinant chimeric 43H7C10 antibody. Expression vectors encodingthe recombinant fully human 43H7C10 antibody and the recombinantchimeric 43H7C10 antibody were transfected by standard techniques intoCHO cells and both forms of the 43H7C10 antibody protein was producedindependently and purified using standard protein purification methods.The two purified antibody proteins were then used in a BIACOREexperiment to determine and compare the affinity to TT, as well ason-rates and off-rates, by standard methodologies. For comparison, 1D6(a fully human anti-TT antibody originally raised in a HuMab Mouse(g)was also produced as a fully human antibody as well as a recombinantchimeric mouse IgG/human kappa antibody (both expressed in CHO cells).Results of the affinity, on-rate and off-rate comparison are shown inTable 7 below.

TABLE 7 Binding Kinetics of Chimeric vs. Human Anti-TT AntibodiesAntibody Affinity On-rate Off-rate Clone Name form (nM) (1/Ms) × 10⁴(1/s) × l0⁴ 43H7C10 Chimeric 17.9 8.9 15.9 43H7C10 Fully human 11.7 4.85.6 1D6 Chimeric 66.9 29.1 19.4 1D6 Fully human 46.3 25.1 11.6

This data demonstrates that a chimeric anti-TT antibody raised in atransgenic mouse of the invention can be recombinantly reconfigured to afully human antibody form and still maintain its binding propertiestoward its target antigen. In fact, the recombinant fully human form ofthe antibody exhibits somewhat higher binding affinity for the targetantigen than the recombinant chimeric form. Furthermore, both thechimeric and fully human recombinant forms of the 43H7C10 antibodyexhibit higher binding affinity for the target antigen than the fullyhuman 1D6 mAb raised in a HuMab Mouse®, thereby demonstrating that thetransgenic mice of the invention can allow for the preparation of afully human antibody against a target of interest that has higheraffinity for the target than a fully human antibody raised in a HuMabMouse®.

1. A transgene construct comprising a plurality of unrearranged human immunoglobulin (Ig) variable region sequences operatively linked to at least one immunoglobulin (Ig) constant region sequence of a non-human host animal, wherein said transgene construct undergoes rearrangement in the non-human host animal and expresses chimeric antibodies in the non-human host animal, the chimeric antibodies comprising a human variable region and a constant region of the non-human host animal.
 2. The construct of claim 1, wherein the plurality of unrearranged human Ig variable region sequences are heavy chain variable region sequences.
 3. The construct of claim 2, wherein the heavy chain variable region sequences comprise V-D-J sequences.
 4. The construct of claim 3, which comprises, in 5′ to 3′ direction, a plurality of human V_(H) regions, a plurality of human D segments, a plurality of human J_(H) segments, a J-μ enhancer from a non-human host animal, a μ switch region from a non-human host animal and a μ constant region from a non-human host animal.
 5. The construct of claim 4, which, when integrated into a non-human host animal genome, undergoes trans-switching with an endogenous constant region of the non-human host animal.
 6. The construct of claim 4, wherein the non-human host animal is a mouse.
 7. The construct of claim 6, which comprises four human V_(H) regions, 15 human D segments and six human J_(H) segments.
 8. The construct of claim 7, which comprises a 9B2 transgene.
 9. The construct of claim 4, which further comprises a γ constant region from a non-human host animal.
 10. The construct of claim 9, wherein the non-human host animal is a mouse.
 11. The construct of claim 4, which comprises all Ig constant regions of the non-human host animal.
 12. The construct of claim 11, wherein the non-human host animal is a mouse.
 13. The construct of claim 1, wherein the unrearranged human Ig variable region sequences are human light chain variable region sequences.
 14. The construct of claim 13, wherein the human light chain variable region sequences are human kappa V-J sequences.
 15. The construct of claim 14, which comprises, in 5′ to 3′ direction, a plurality of human V_(κ) regions, a plurality of human J_(κ) segments, a J-κ enhancer from a non-human host animal and a C_(κ) coding region from a non-human host animal.
 16. The construct of claim 15, wherein the non-human host animal is a mouse.
 17. A trans gene construct which comprises, in 5′ to 3′ direction, a plurality of human V_(H) regions, a plurality of human D segments, a plurality of human J_(H) segments, a mouse J-μ enhancer, a mouse μ switch region and a mouse μ constant region, wherein the transgene construct, when integrated into a mouse genome, undergoes trans-switching with an endogenous mouse γ constant region such that chimeric antibodies comprising human V regions and mouse constant regions of IgM and IgG isotype are produced in the mouse.
 18. A transgenic non-human host animal comprising the transgene construct of claim 1, wherein the animal expresses chimeric antibodies comprising human Ig variable regions and non-human host animal Ig constant regions. 19.-24. (canceled)
 25. A method of making a chimeric antibody specific for an antigen of interest comprising immunizing the transgenic non-human host animal of claim 18 with the antigen of interest and obtaining from the animal a chimeric antibody specific for the antigen of interest.
 26. The method of claim 25, further comprising isolating from the animal a nucleic acid encoding the chimeric antibody and replacing nucleic acid encoding the non-human host animal Ig constant region with nucleic acid encoding a human Ig constant region to thereby convert the chimeric antibody to a human antibody and expressing the human antibody.
 27. (canceled) 